Collagen is the most abundant protein in the body, presenting many biological signals and maintaining the mechanical integrity of many different tissues. Its molecular organization determines its function, which has made collagen fibrillogenesis a topic of interest in many research fields. Collagen has the ability to self-associate in vitro, forming gels that can act as a 3-dimensional substrate, and provide mechanical and biological signals for cell growth. Research on collagen fibrillogenesis with and without additional extracellular matrix components has raised many questions about the interplay between collagen and other extracellular matrix molecules. There are more than 20 types of collagen currently identified, with type I being the most common. Many tissues are composed primarily of type I collagen including tendon, ligament, skin, and bone. While each of these structures also contains other collagen types, proteoglycans and glycosaminoglycans, and minerals in the case of bone, the principle component is type I collagen. The dramatic difference in mechanical integrity each of these structures exhibits is largely due to the intricate organization of collagen and the interplay with other non-collagen type I components.
Decorin is a proteoglycan that is known to influence collagen fibrillogenesis, which consequently can modify the mechanical and biological information in a collagen gel. The signals resulting from structural changes in collagen organization, as well as the unique signals contained in the glycosaminoglycan chains that are part of proteoglycans, alter cellular behavior and offer a mechanism to design collagen matrices to provide desired cellular responses. Consequently, we have developed collagen-binding synthetic peptidoglycans which influence collagen organization at the molecular level. These collagen-binding synthetic peptidoglycans are designed based on collagen binding peptides attached to, for example, a glycan, such as a glycosaminoglycan or a polysaccharide, and can be tailored with respect to these components for specific applications. The collagen-binding synthetic peptidoglycans described herein influence the morphological, mechanical, and biological characteristics of collagen matrices, and consequently alter cellular behavior, making these molecules useful for tissue engineering applications.
In one embodiment, an engineered collagen matrix comprising a collagen matrix and a collagen-binding synthetic peptidoglycan is provided. In this embodiment, the 1) collagen can be crosslinked or uncrosslinked, 2) the collagen-binding synthetic peptidoglycan can have amino acid homology with a portion of the amino acid sequence of a protein or a proteoglycan that regulates collagen fibrillogenesis or the collagen-binding synthetic peptidoglycan can be an aberrant collagen-binding synthetic peptidoglycan, 3) the engineered collagen matrix can further comprise an exogenous population of cells, 4) the exogenous population of cells can be selected from non-keratinized or keratinized epithelial cells or a population of cells selected from the group consisting of endothelial cells, mesodermally derived cells, mesothelial cells, synoviocytes, neural cells, glial cells, osteoblast cells, fibroblasts, chondrocytes, tenocytes, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, multi-potential progenitor cells (e.g., stem cells, including bone marrow progenitor cells), and osteogenic cells, 5) the engineered collagen matrix can further comprise at least one polysaccharide, 6) the collagen-binding synthetic peptidoglycan can be a compound of formula PnGx wherein n is 1 to 10, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, and wherein G is a glycan (e.g. a glycosaminoglycan or a polysaccharide), 7) the collagen-binding synthetic peptidoglycan can be a compound of formula (PnL)xG wherein n is 1 to 5, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, wherein L is a linker, and wherein G is a glycan, 8) the collagen-binding synthetic peptidoglycan can be a compound of formula P(LGn)x wherein n is 1 to 5, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, wherein L is a linker, and wherein G is a glycan, 9) the synthetic peptide can have amino acid homology with the amino acid sequence of a small leucine-rich proteoglycan or a platelet receptor sequence, 10) the synthetic peptide can have amino acid homology with the amino acid sequence of a platelet collagen receptor sequence, 11) the peptide can comprise an amino acid sequence selected from the group consisting of RRANAALKAGELYKSILYGC [SEQ ID NO: 1], RLDGNEIKRGC [SEQ ID NO: 2], AHEEISTTNEGVMGC [SEQ ID NO: 3], NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ ID NO: 4], CQDSETRTFY [SEQ ID NO: 5], TKKTLRTGC [SEQ ID NO: 6], GLRSKSKKFRRPDIQYPDATDEDITSHMGC [SEQ ID NO: 7], SQNPVQPGC [SEQ ID NO: 8], SYIRIADTNITGC [SEQ ID NO: 9], SYIRIADTNIT [SEQ ID NO: 10], KELNLVYT [SEQ ID NO: 11], KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV [SEQ ID NO: 13], and GSITTIDVPWNVGC [SEQ ID NO: 14], 12) the glycan can be selected from the group consisting of alginate, agarose, dextran, chondroitin, dermatan, dermatan sulfate, heparan, heparin, keratin, and hyaluronan, 13) the glycan can be selected from the group consisting of dermatan sulfate, dextran, and heparin, 14) the collagen can be selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, and combinations thereof, 15) the glycan can be a glycosaminoglycan or a polysaccharide, or 16) the invention can include any combination of the features described in this paragraph.
In another illustrative embodiment, a method of preparing an engineered collagen matrix is provided. The method comprises the steps of providing a collagen solution, providing a collagen-binding synthetic peptidoglycan, and polymerizing the collagen in the presence of the collagen-binding synthetic peptidoglycan to form the engineered collagen matrix. This embodiment can include any of the features described in the preceding paragraph. Also, in this embodiment, the amount of collagen in the collagen solution can be from about 0.4 mg/mL to about 6 mg/mL, and the molar ratio of the collagen to the collagen-binding synthetic peptidoglycan can be from about 1:1 to about 40:1.
In yet another embodiment a compound of formula PnGx is provided wherein n is 1 to 10, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, and wherein G is a glycan.
In a further embodiment, a compound is provided of formula (PnL)xG wherein n is 1 to 5, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, wherein L is a linker, and G is a glycan.
In still another illustrative embodiment, a compound is provided of formula P(LGn)x wherein n is 1 to 5, wherein x is 1 to 10, wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of a collagen-binding domain, wherein L is a linker, and wherein G is a glycan. In any of these compound embodiments the linker can comprise the formula —SCH2CH2C(O)NHN═, the glycan can be a glycosaminoglycan or a polysaccharide, and any applicable features described above can also be included.
In another aspect, a method of altering the structure or mechanical characteristics of an engineered collagen matrix is provided. The method comprises the steps of providing a collagen solution, providing a collagen-binding synthetic peptidoglycan, and polymerizing the collagen in the presence of the collagen-binding synthetic peptidoglycan to form the altered, engineered collagen matrix. Any applicable features described above can also be included.
In another embodiment, a kit is provided. The kit can comprise any of the engineered collagen matrices described above. In this embodiment, the engineered collagen matrix can be sterilized, and the kit can further comprise cells wherein the cells can be selected from the group consisting of mesothelial cells, synoviocytes, progenitor cells, fibroblasts, neural cells, glial cells, osteoblast cells, chondrocytes, tenocytes, endothelial cells, and smooth muscle cells. The engineered collagen matrix can comprise any of the compounds described above.
In one embodiment, a method for inhibiting activation of platelets is described, the method comprising the step of providing a collagen-binding synthetic peptidoglycan for contacting collagen wherein the collagen-binding synthetic peptidoglycan binds to the collagen and wherein activation of the platelets is inhibited. In another embodiment, a method for inhibiting adhesion of platelets to collagen is described, the method comprising the step of providing a collagen-binding synthetic peptidoglycan for contacting collagen wherein the collagen-binding synthetic peptidoglycan binds to the collagen, and wherein adhesion of the platelets to collagen is inhibited. In another embodiment, either of the above methods wherein the glycan is selected from the group consisting of hyaluronan, heparin, and dextran is provided. In still another embodiment, the collagen-binding synthetic peptidoglycan used in any of the above methods comprises a peptide selected from the group consisting of RRANAALKAGELYKSILYGC [SEQ ID NO: 1], GSITTIDVPWNV [SEQ ID NO: 13], and GSITTIDVPWNVGC [SEQ ID NO: 14].
In yet another embodiment, a graft construct is provided. The graft construct comprises any of the engineered collagen matrices described above.